Clin Biochem, Vol. 25, pp. 387-394, 1992 Printed in the USA. All rights reserved.

0009-9120/92 $5.00 + .00 Copyright © 1992 The Canadian Society of Clinical Chemists.

Effect of Cyclosporin on Plasma Lipoprotein Lipase Activity in Rats JOSE LOPEZ-MIRANDA, 1 FRANCISCO PCREZ-JIMI~NEZ, 1 JUAN A. GOMEZ-GERIQUE, ANTONIO ESPINO-MONTORO, 1 LUIS HIDALGO-ROJAS, 1, JAVIER PEDRENO, 2 and JOSE A. JIMI~NEZ-PEREPI~REZ 1 1Unidad de Lipidos y Arteriosclerosis, Hopital Universitario Reina Sofia, Avda. Men~ndez Pidal, s/n. 14004 Cordoba and 2Servicio de Bioquimica, Hospital de la Santa Cruz y San Pablo, San Antonio Maria Claret, 167, 08025 Barcelona, Spain The effects of cyclosporin on plasma lipoproteins and lipoprotein lipase (LPL) activity were studied in rats treated with different doses of the drug for periods ranging between 7 and 30 days. The treatment with cyclosporin resulted in an increase in plasma triglycerides and non-HDL-cholesterol, and a dose and time-dependent decrease of LPL activity and HDL-cholesterol, mainly because of a fall in the HDL2-cholesterol subfraction. The decrease of LPL activity was positively correlated (p < 0.01 ) with plasma HDL-cholesterol and HDL2-cholesterol and negatively with plasma triglycerides and non-HDL-cholesterol (p < 0.01). Our results indicate that the decrease in plasma LPL activity may be responsible for the increase in plasma triglycerides and the decrease in plasma HDL-cholesterol found in rats under cyclosporin treatment.

KEY WORDS: cyclosporin; lipoproteins; lipoprotein lipase; cholesterol; triglycerides; HDL-cholesterol. Introduction 'ow that new technologies can prevent and manage acute rejection, accelerated atherosclerosis N has become a major obstacle to the long-term survival of heart transplant recipients (1). Coronary artery disease (CAD) is present in up to 40% of patients by the third year post-transplantation, and accounts for about 50% of graft failures beyond 2 years after transplantation (2,3). Furthermore, an increase of total serum cholesterol and triglycerides has been found 6 months after heart transplantation, mainly due to an increase in LDL cholesterol (4-8). A chronic form of graft rejection is thought to play a central role in the development of this form of atherosclerosis (9). It has been suggested that hyperlipidemia after heart transplantation may be a causative factor in these events (9-11). Several studies have shown that cyclosporin may produce an elevation of total serum cholesterol and triglycerides, due to an increase in LDL and VLDL (12-16).

Correspondence: Prof. Francisco P~rez-Jim~nez. Manuscript received April 2, 1992; revised June 6, 1992; accepted July 8, 1992. CLINICAL

BIOCHEMISTRY,

VOLUME

25, OCTOBER

1992

However, the mechanism by which cyclosporin induces these changes remains to be determined. Recently, a decrease in lipoprotein lipase (LPL) activity in kidney and heart transplant patients has been shown, which was inversely correlated with plasma triglycerides and positively correlated with HDL-cholesterol and HDL2-cholesterol (17,18). However, attempts to define the mechanisms involved are confounded by dramatic changes in metabolism and nutritional status after transplantation, as well as by treatment with multiple immunosuppressive and antihypertensive drugs that can modify lipoprotein metabolism and LPL activity. It has also been reported that cyclosporin exerts a direct cytotoxic effect on endothelial cells (19). In this study we have examined the effect of cyclosporin on LPL activity in rats to determine whether changes in its activity can explain the increase in triglycerides and the decrease in HDL-cholesterol found in animals and humans treated with cyclosporin. Materials and methods ANIMALS

AND EXPERIMENTAL

PROCEDURES

Male Wistar rats (n = 109), weighing 250-300 g, were divided into 15 groups of similar weight, and housed in individual metabolic cages. Three groups received cyclosporin (CyA) parenterally at doses of 15, 30, or 50 mg/kg/day for a week, and two other groups were treated for a month at doses of 15 and 30 mg/kg/day (Cyclosporin rats). These rats received an ad libitum liquid diet (Panlab, S.A., Barcelona, Spain) that simplified the recording of the daily food consumption for the purpose of pair feeding. Another five groups of rats (Excipient rats) received the excipient of the parenteral cyclosporin (Cremaphor EL, Sigma Co., St Louis, MO, USA) during the same time and at the same dosages as the CyA-treated rats. Finally, five other groups were used as a second control (Untreated rats). The excipient and untreated groups were introduced in the study 1 day later, and they were fed each day with the same 387

LOPEZ-MIRA.NDA ETAL.

number of calories ingested by their paired CyAtreated rats the previous day. All had free access to water. After a 12-h fast, the animals were anesthetized with sodium pentobarbital (12 mg/kg i.p.) and 1 mL of blood was obtained by venous puncture and pooled in tubes containing EDTA (1 mg/mL) for measurement of total cholesterol, triglycerides, and HDLcholesterol. The apo B-containing lipoproteins (VLDL, LDL, and intermediate-density lipoprot e i n s ) c h o l e s t e r o l ( r e f e r r e d to as n o n - H D L cholesterol) was calculated as the difference bet w e e n total cholesterol a n d HDL-cholesterol. Plasma was obtained from blood drawn by cardiac puncture 15 min after intravenous administration of 100 USP units of heparin into the femoral vein. Duplicate samples were drawn in evacuated tubes containing heparin as anticoagulant and immediately placed on ice. Plasma was separated by centrifugation at 4 °C, and stored at - 4 0 °C. PLASMA LIPID ASSAYS

Cholesterol and triglycerides in plasma were d e t e r m i n e d by s t a n d a r d enzymatic procedures (Boehringer Mannheim, Germany). HDL and the subfractions HDL2 and HDL3 were separated by the precipitation methods of Gidez et al. (20). Cholesterol was determined in the supernatant after precipitation. ASSAYS FOR LPL ACTIVITIES

The standard assay was carried out using Corey and Zilversmit's method (21). Substrates were prepared by mixing 12.5 ~Ci of glycerol-tri-(1-14C)oleate (Amersham, UK), 69 mg of trioleoyl glycerol, 6 mg of soybean lecithin, and 5 mL of glycerol. The mixture was homogenized with a 70-W MSE ultrasonic disintegrator (model MK2, Measuring Scientific Equipment, UK). A 5% (w/v) solution of h u m a n albumin was prepared in 0.15 M NaC1, 0.2 M Tris, pH 8.2. This solution was combined with heated rat plasma and the stock glycerol emulsion in proportions of 1:0.5:1 (v/v/v) respectively. For the assay, 0.05 mL of 0.15 M NaC1 was combined with 0.05 mL of postheparin plasma and 0.100 mL of emulsion in a glass tube. All assays were terminated by the addition of 3.5 mL of the solvent system of Belfrage and Vaughan (22). Free fatty acids were extracted by the addition of 1 mL of 0.1 M potassium carbonate-potassium borate buffer, pH 10.5 (23). The radioactivity in a 0.6 mL aliquot of the upper phase was determined by liquid scintillation counting in Bray's solution (24). The enzymatic activity is expressed as mU, defined as the nanomoles of labeled fatty acids liberated per mL of enzyme source per min. STATISTICAL METHODS

The results obtained are expressed as the mean +SEM, and were analyzed by the one-way analysis of 388

variance (ANOVA) followed by the N e u m a n s Keuls test. It was previously demonstrated that data followed a normal distribution by the KolmogorovSmirnov test. Correlations between lipid concentrations and LPL activity were determined using Pearson's product moment coefficient, p values < 0.05 were considered significant. Results

No statistically significant changes in body weight among any of the groups occurred during the experiments (data not shown). LIPID STUDIES Mean plasma cholesterol, triglycerides, HDLcholesterol, HDL3-cholesterol, a n d non-HDLcholesterol in untreated, excipient, and cyclosporintreated rats, are shown in Tables i and 2. Total cholesterol increased in animals treated with low doses of cyclosporin for 1 week. No differences were observed in other groups treated with cyclosporin at other doses and times. However, the cyclosporintreated rats showed an increase in plasma triglycerides and non-HDL-cholesterol, and a dose- and time-dependent decrease of plasma HDL-cholesterol due to a fall in the HDL2-cholesterol subfraction (Figure 1). L P L ACTIVITIES

Figure 2 shows the activities of plasma LPL in untreated rats, excipient, and cyclosporin-treated rats during 1 week and 1 month, respectively. Cyclosporin caused a dose- and time-dependent decrease of plasma LPL activity. Significant positive correlations were found between plasma LPL activity a n d p l a s m a H D L - c h o l e s t e r o l a n d HDL 2cholesterol (Figure 3). Plasma triglycerides and nonHDL cholesterol were negatively correlated with plasma LPL activity (Figure 3). Discussion

The present study was performed to evaluate the role of LPL in the alterations of plasma lipoproteins induced by cyclosporin in rats. We found that the drug increases plasma triglycerides and non-HDLcholesterol, and decreases plasma HDL-cholesterol. Furthermore, cyclosporin induced a dose- and timedependent decrease of plasma LPL activity positively correlated with the decrease of plasma HDL cholesterol and HDL2-cholesterol and negatively correlated with plasma triglycerides and non-HDL cholesterol. These findings may explain the decrease in plasma HDL2-cholesterol induced by cyclosporin, since the transformation of HDL 3 particles to HDL2 takes place with the hydrolysis of triglyceride-rich lipoproteins (25). The increase observed in total cholesterol in the group treated for 1 week with the lower doses of cyclosporin may be due to the fact that cyclosporin CLINICALBIOCHEMISTRY,VOLUME25, OCTOBER1992

CYCLOSPORIN A N D LIPOPROTEIN LIPASE TABLE 1

Total Cholesterol, Triglycerides, HDL-Cholesterol (HDL-C), HDL3-Cholesterol (HDL3-C), and Cholesterol in apo B-Containing Lipoproteins (non-HDL-C) in Cyclosporin, Excipient, and Untreated Rats After 1 Week Cyclosporin 15 mg/kg/day [7) Cholesterol Triglycerides HCL-C HCL3-C Non-HDL-C 30 mg/kg/day [7] Cholesterol Triglycerides HCL-C HDL3-C Non-HDL-C 50 mg/kg/day [8] Cholesterol Triglycerides HDL-C HDL3-C Non-HDL-C

Excipient a

Untreated

2.46 0.64 1.42 0.91 1.04

-+ 0.05 b ± 0.06 b ± 0.1 ± 0.05 ± 0.07 b

1.94 0.42 1.37 0.75 0.57

-+ 0.05 ± 0.01 ± 0.05 -+ 0.08 ± 0.05 c

2.09 0.38 1.29 0.70 0.80

-+ 0.1 ± 0.03 ± 0.1 ± 0.05 ± 0.03

1.84 0.58 1.09 0.78 0.75

± ± ± ± ±

0.13 0.1 b 0.1 b 0.05 0.03 b

2.02 0.38 1.50 0.88 0.52

± 0.12 -+ 0.06 ± 0.1 ± 0.05 ± 0.08

1.84 0.29 1.32 0.75 0.52

± ± ± ± ±

1.89 0.96 0.88 0.67 1.01

± ± ± ± +

0.08 0.16 b 0.1 b 0.08 0.08 b

1.99 0.30 1.53 0.91 0.46

+- 0.08 ± 0.02 -+ 0.08 -+ 0.07 ± 0.03

1.89 0.35 1.32 0.83 0.57

-+ 0.16 ± 0.04 +- 0.1 - 0.05 ± 0.06

0.16 0.02 0.12 0.08 0.05

Number of subjects in brackets. aResults are mM/L (mean +- SEM). bp < 0.05 between cyclosporin and excipient groups. Cp < 0.05 between excipient and untreated groups. induced a n i n c r e a s e in n o n - H D L - c h o l e s t e r o l in all g r o u p s , w i t h no c h a n g e s in H D L - c h o l e s t e r o l in a n i m a l s t r e a t e d w i t h t h e lower doses. C o n s e q u e n t ly, t h e effect of h i g h cyclosporin doses on HDL-cholesterol b l u n t e d the expected increase of total cholesterol. A l t h o u g h it is difficult to e x t r a p o l a t e t h e s e findings in r a t s to c y c l o s p o r i n - t r e a t e d h u m a n s , several studies on h e a r t t r a n s p l a n t p a t i e n t s ( 5 - 8 ) a n d cy-

c l o s p o r i n - t r e a t e d p a t i e n t s ( 1 2 - 1 5 ) h a v e s h o w n similar plasma LDL-cholesterol and triglyceride c h a n g e s to those r e p o r t e d here. H o w e v e r , m a n y of t h e s e h a v e not s h o w n a decrease of p l a s m a H D L cholesterol. T h e s e differences m a y be due to t h e fact t h a t t h e p a t i e n t s i n c l u d e d in t h e s e s t u d i e s w e r e t r e a t e d w i t h p r e d n i s o n e a n d a z a t h i o p r i n e , besides cyclosporin. It is k n o w n t h a t corticosteroid t r e a t m e n t m a y induce a n i n c r e a s e in p l a s m a H D L - c h o -

TABLE 2 Total Cholesterol, Triglycerides, HDL-Cholesterol (HDL-C), HDLz-Cholesterol (HDLz- C), and Cholesterol in apo B-Containing Lipoproteins (Non-HDL-C) in Cyclosporin, Excipient, and Untreated Rats After 1 Month

15 mg/kg/day [7] Cholesterol Triglycerides HDL-C HDL3-C Non-HDL-C 30 mg/kg/day [7] Cholesterol Triglycerides HDL-C HDL3-C Non-HDL-C

Cyclosporin"

Excipient"

Untreated ~

1.91 0.79 1.06 0.72 0.75

± ± ± ± -

0.1 0.06* 0.05* 0.05 0.08*

1.97 0.32 1.45 0.80 0.52

- 0.16 - 0.02 -+ 0.09 ± 0.05 ± 0.04

1.78 0.34 1.34 0.78 0.44

± 0.08 ± 0.03 ± 0.03 ± 0.02 +- 0.03

1.91 0.79 1.06 0.75 0.85

_+ 0.08 ~- 0.09* -+ 0.02* -+ 0.03 ± 0.08*

1.91 0.32 1.40 0.80 0.51

± 0.07 ± 0.03 ± 0.05 -+ 0.05 +- 0.04

1.89 0.38 1.40 0.78 0.49

-+ 0.1 -+ 0.05 +- 0.04 -+ 0.06 ± 0.05

Number of subjects in brackets. aResults are mM/L (mean _+ SEM). bp < 0.05 between cyclosporin and excipient groups. CLINICAL BIOCHEMISTRY, VOLUME 25, OCTOBER 1992

389

LOPEZ-MIRANDAETAL.

mmol/L A

15 m g / k g / d a y

50 m g / k g / d a y

30 m g / k g / d a y

mmol/L B 0.8

1 0.6

.

1

1

T

:i i:i!i

0.4

0.3

15 m g / k g / d a y CYCLOSPORINRATS ~

30 m g / k g / d a y EXCIPIENTRATS

[

[ UNTREATEDRATS

Figure 1 - - Plasma HDL2-cholesterol (HDL2-C) concentrations of untreated, excipient, and cyclosporin-treated rats after 1 week (A) and one month (B). Results are the mean -+ SEM (*p < 0.05 comparing cyclosporin-treated rats with both control groups). lesterol and in LPL activity (26,27). In agreement with this hypothesis, Taylor et al. (28) showed t h a t plasma HDL-cholesterol from heart-transplant patients treated with cyclosporin without corticosteroid were lower t h a n in patients treated with pred390

nisone and cyclosporin. Furthermore, Luke et al. (15) f o u n d t h a t c y c l o s p o r i n d e c r e a s e s H D L cholesterol in b o n e - m a r r o w t r a n s p l a n t p a t i e n t s treated only with cyclosporin. Decreases in plasma LPL activity as well as an increase in plasma triCLINICALBIOCHEMISTRY,VOLUME 25, OCTOBER 1992

CYCLOSPORIN AND LIPOPROTEIN LIPASE

500

mU A

400

300

T

T

T

15 m g / k g / d a y

30 m g / k g / d a y

H

300

100

500

50 m g / k g / d a y

mU

400

T

T

*

T

300

200

100

0

15mg/kg/day

CYCLOSPORIN RATS

30rng/kg/day

~

EXCIPIENT RATS

[

] UNTREATED RATS

Figure 2 - - Plasma LPL activities of untreated, excipient, and cyclosporin-treated rats after 1 week (A) and one month (B). Results are the mean +- SEM (*p < 0.05 comparing cyclosporin-treated rats with both control groups).

glycerides have been reported by Derfler et al. (17) a n d S u p e r k o et al. (18) in k i d n e y a n d h e a r t transplant patients, respectively. In the latter study (18) the decrease in LPL activity was inversely correlated with triglyceride concentrations and cyclosporin doses, and positively with HDL-cholesterol CLINICALBIOCHEMISTRY,VOLUME 25, OCTOBER 1992

and HDL2-cholesterol. Low HDL2-cholesterol concentrations were reported by Ettinger et al. (29) in renal-transplant recipients following cyclosporin treatment. Despite m a n y studies, attempts to define the mechanism for hyperlipidemia in transplant recipients are confounded by dramatic changes in me391

L O P E Z - M I R A N D A ET AL.

500 -

400

LPL ( m U )

500

LPL ( m U )

400

-



.

300 -

300 • •

&



200 -

200

I00

i00

-

r = 0.439 p

Effect of cyclosporin on plasma lipoprotein lipase activity in rats.

The effects of cyclosporin on plasma lipoproteins and lipoprotein lipase (LPL) activity were studied in rats treated with different doses of the drug ...
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